Method for detecting transferrin receptor-like proteins

ABSTRACT

The present invention provides isolated nucleic acids encoding TfR2 polypeptides, or fragments thereof, and isolated TfR2 polypeptides encoded thereby. Further provided are vectors containing the nucleic acids of the present invention, host cells transformed therewith, antisense oligonucleotides thereto and compositions containing antibodies that specifically bind to invention polypeptides. Methods of detecting TfR2 protein in a cell are also provided.

RELATED APPLICATION

This application claims the benefit of a provisional application Ser.No. 60/107,502, filed on Nov. 6, 1998 which is hereby incorporated byreference.

This invention was made with support by Grant No. CA26038-20 awarded bythe National Institutes of Health.

BACKGROUND OF THE INVENTION

1. Area of the Art

The invention relates generally to the transferrin receptor family andspecifically to nucleic acid encoding transferrin receptor-likeproteins, and products related thereto.

2. Description of the Prior Art

Throughout this application, various references are referred to withinparentheses. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citation for these references may be found at the end ofthis application, preceding the claims. In addition, the abbreviationsused are: TfR, transferrin receptor; RT-PCR, reversetranscriptase-polymerase chain reaction; Tf, transferrin; PSMA, prostatespecific membrane antigen; RACE, rapid amplification of cDNA ends;G3PDH, glyceraldehyde-3-phosphate dehydrogenase; UTR, untranslatedregion; IRE, iron—responsive element; IRP, iron regulatory protein.

Transferrin receptor (TfR) is a key molecule involved in iron uptake bycells (1, 2). On the cell membrane the TfR homodimer binds to twodiferric transferrin (Tf) molecules, resulting in internalization of thecomplex. In the cytoplasm, iron is released and utilized as a co-factorby several proteins, including heme, aconitase, cytochromes (3) andribonucleotide reductase (4), or it may be stored in ferritin molecules.Since dividing cells require more iron than non-dividing cells, theexpression of TfR is usually higher in rapidly dividing tissue (5), suchas hematopoietic progenitor cells (6). Also, TfR expression is higher intumor cells when compared to their normal cellular counterparts (7). Theaffinity of diferric Tf to TfR is modulated by HFE (8, 9).

The only other known homolog of TfR is PSMA, a human homolog of murineNAAG-peptidase (10, 11). Since the expression of PSMA is high inprostate cancer, the antibody against PSMA was approved for use as animaging agent to detect metastasis of prostate cancer (12). The functionof PSMA appears to be considerably different from that of TfR, despitethe modest similarity between their extracellular domains. PSMA does notmediate endocytosis, and possesses glutamyl-carboxypeptidase activity(11, 13).

Given the importance of a transferrin receptor in an iron uptake processof cells, it is desirable to identify potential molecules which arehomologous to a transferrin receptor and which perform transferrinreceptor-like functions. The identification of the molecules may providevaluable tools for altering the iron uptake of specific cells. Inaddition, identified novel receptors may be used to identify various newligand that have activity with other metals or other key proteins thatare vital for the cells. Furthermore, since TfR expression is higher intumor cells, the newly identified receptors may be used for diagnosingor treating tumor cells.

SUMMARY OF THE INVENTION

It is an object of the present invention to identify the potentialtransferrin receptor-like proteins. It is also an object of the presentinvention to investigate the roles of newly discovered receptors in ironmetabolism in cells. It is a further object of the present invention toprovide methods for diagnosing tumor cells.

Accordingly, the present invention provides isolated nucleic acidsencoding novel transferrin receptor-like (TfR2) polypeptides, orfragments thereof, and isolated TfR2 polypeptides encoded thereby.Further provided are vectors containing nucleic acids of the presentinvention, host cells transformed therewith, antisense oligonucleotidesthereto and compositions containing antibodies that specifically bind topolypeptides of the present invention. Methods of detecting TfR2 in acell are also provided.

The invention is defined in its fullest scope in the appended claims andis described below in its preferred embodiments.

DESCRIPTION OF THE FIGURES

The above-mentioned and other features of this invention and the mannerof obtaining them will become more apparent and will be best understoodby reference to the following description, taken in conjunction with theaccompanying drawings. These drawings depict only a typical embodimentof the invention and do not therefore limit its scope. They serve to addspecificity and detail, in which:

FIG. 1 is a gene map of the transferrin receptor 2 (TfR2) gene.

FIG. 2 shows DNA sequences of exons 3-5 of TfR2 gene. Boxed sequenceswere found only in the β transcript.

FIG. 3 shows deduced amino acid sequence of TfR2-α, aligned with thosefor the human TfR and PSMA proteins.

FIGS. 4A and 4B show the results of Northern blot analysis on multipletissue blots of human mRNA (A), and cell line blots of total RNA (B).

FIGS. 5A and 5B show the representative results of RT-PCR analysesperformed with primers for α and β transcripts of TfR2 (35 cycles) aswell as G3PDH (27 cycles).

FIGS. 6A, 6B and 6C show the expression and functional analysis ofTfR2-α protein.

FIG. 7 is the amino acid sequence of TfR2 protein SEQ ID NO:1.

FIG. 8 is the DNA sequence of TfR2-α gene SEQ ID NO:2.

FIG. 9 is the DNA sequence of TfR2-β gene SEQ ID NO:3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery and the cloning of ahuman gene homologous to transferrin receptor (TfR). For the purpose ofthe present invention, this gene is termed TfR2 gene. TfR2 gene iscloned, sequenced and mapped to chromosome 7q22. Two transcriptsexpressed from this gene are identified; they are α (about 2.9 kb) and β(about 2.5 kb) transcripts. The deduced amino acid sequences from eachtranscript predict the possible expression of both a membrane bound andan intracellular form of the TfR2 protein. The deduced amino acidsequence of TfR2-α protein is a type II membrane protein, and shares 45%identity and 66% similarity in its extracellular domain with TfR. TheTfR2-β protein lacked the amino terminal protein of the TfR2-α proteinincluding the putative transmembrane domain. TfR deficient cellstransfected with FLAG-tagged TfR2-α showed an increase of biotinylatedtransferrin (Tf) binding to the cell surface. In addition, thesetransfected cells have a marked increase of Tf-bound ⁵⁵Fe uptake.

Accordingly, the present invention provides isolated nucleic acidsencoding a TfR2 polypeptide. Such nucleic acids can be obtained, forexample, from human chromosome 7q22. Deletion or loss of heterozygosityof this chromosomal region has been reported in several malignantdiseases including myelodysplastic syndromes, acute myeloid leukemia, aswell as breast, ovarian and pancreatic cancers. The nucleic acids mayalso be obtained from a human cDNA library such as, but not limited to,HL60 cDNA library or TF-1 cDNA library.

The term “nucleic acids” (also referred to as polynucleotides) refers toa polymer of deoxyribonucleotides or ribonucleotides, in the form of aseparate fragment or as a component of a larger construction. DNAencoding the polypeptide of the invention can be assembled from cDNAfragments or from oligonucleotides which provide a synthetic gene whichis capable of being expressed in a recombinant transcriptional unit.Polynucleotide sequences of the invention include DNA, RNA and cDNAsequences. In accordance with one embodiment of the present invention,nucleic acids encode a polypeptide having the amino acid sequence setforth in SEQ ID NO:1 (see FIG. 7). In accordance with another embodimentof the present invention, nucleic acids may include, but are not limitedto, nucleic acids having substantially the same nucleotide sequence asnucleotides set forth in SEQ ID NO: 2 or SEQ ID NO:3 (FIG. 8 and FIG. 9,respectively). In accordance with a preferred embodiment, the nucleicacids of the present invention include the same nucleotide sequences asset forth in the SEQ ID NO:2 or 3. As used herein, the phrase“substantially the same nucleotide sequence” refers to DNA havingsufficient homology to the reference polynucleotide, such that it willhybridize to the reference nucleotide under typical moderate stringencyconditions. DNA having “substantially the same nucleotide sequence” asthe reference nucleotide sequence has at least 60% homology with respectto the reference nucleotide sequence.

As used herein, the phrase “isolated” means a nucleic acid that is in aform that does not occur in nature. DNA sequences of the invention canbe obtained by several methods. For example, the DNA can be isolatedusing hybridization techniques which are well known in the art. Theseinclude, but are not limited to: 1) hybridization of genomic or cDNAlibraries with probes to detect homologous nucleotide sequences, 2)polymerase chain reaction (PCR) on genomic DNA or cDNA using primerscapable of annealing to the DNA sequence of interest, and 3) antibodyscreening of expression libraries to detect cloned DNA fragments withshared structural features.

Preferably the polynucleotide of the invention is derived from amammalian organism, and most preferably from a mouse, rat, or human.Screening procedures which rely on nucleic acid hybridization make itpossible to isolate any gene sequence from any organism, provided theappropriate probe is available. Oligonucleotide probes, which correspondto a part of the sequence encoding the protein in question, can besynthesized chemically. This requires that short, oligopeptide stretchesof an amino acid sequence must be known. The DNA sequence encoding theprotein can be deduced from the genetic code, however, the degeneracy ofthe code must be taken into account. It is possible to perform a mixedaddition reaction when the sequence is degenerate. This includes aheterogeneous mixture of denatured double-stranded DNA. For suchscreening, hybridization is preferably performed on eithersingle-stranded DNA or denatured double-stranded DNA. Hybridization isparticularly useful in the detection of cDNA clones derived from sourceswhere an extremely low amount of mRNA sequences relating to thepolypeptide of interest are present. In other words, by using stringenthybridization conditions directed to avoid non-specific binding, it ispossible, for example, to allow the autoradiographic visualization of aspecific cDNA clone by the hybridization of the target DNA to thatsingle probe in the mixture which is its complete complement (Wallace,et al., Nucl. Acid Res., 9:879, 1981).

The specific DNA sequences of the present invention can also be obtainedby: 1) isolation of double-stranded DNA sequences from the genomic DNA;2) chemical manufacture of a DNA sequence to provide the necessarycodons for the polypeptide of interest; and 3) in vitro synthesis of adouble-stranded DNA sequence by reverse transcription of mRNA isolatedfrom an eukaryotic donor cell. In the latter case, a double-stranded DNAsequence by reverse transcription of mRNA isolated from an eukaryoticdonor cell. In the latter case, a double-stranded DNA complement of mRNAis eventually formed which is generally referred to as cDNA. Of thethree above-noted methods for developing specific DNA sequences for usein recombinant procedures, the isolation of genomic DNA isolates is theleast common. This is especially true when it is desirable to obtain themicrobial expression of mammalian polypeptides due to the presence ofintrons. The synthesis of DNA sequences is frequently the method ofchoice when the entire sequence of amino acid residues of the desiredpolypeptide product is known. When the entire sequence of amino acidresidues of the desired polypeptide is not known, the direct synthesisof DNA sequences is not possible and the method of choice is thesynthesis of cDNA sequences. Among the standard procedures for isolatingcDNA sequences of interest is the formation of plasmid or phage-carryingcDNA libraries which are derived from reverse transcription of mRNA,which is abundant in donor cells that have a high level of geneticexpression. When used in combination with polymerase chain reactiontechnology, even rare expression products can be cloned. In those caseswhere significant portions of the amino acid sequence of the polypeptideare known, the production of labeled single or double-stranded DNA orRNA probe sequences duplicating a sequence putatively present in thetarget cDNA may be employed in DNA/DNA hybridization procedures whichare carried out on cloned copies of the cDNA which have been denaturedinto a single-stranded form (Jay, et al., Nucl. Acid Res., 11:2325,1983).

DNA sequences encoding TfR2 polypeptides can be expressed in vitro byDNA transfer into a suitable host cell. “Host cells” are cells in whicha vector can be propagated and its DNA expressed. The term also includesany progeny of the subject host cell. It is understood that all progenymay not be identical to the parental cell, since there may be mutationsthat occur during replication. However, such progeny are included whenthe term “host cell” is used. Methods of stable transfer, meaning thatthe foreign DNA is continuously maintained in the host, are known in theart.

In the present invention, the polynucleotide sequences may be insertedinto a recombinant expression vector. The term “recombinant expressionvector” refers to a plasmid, virus or other vehicle known in the artthat has been manipulated by insertion of incorporation of the TfR2genetic sequences. Such expression vectors contain a promoter sequencewhich facilitates the efficient transcription of the inserted geneticsequence of the host. The expression vector typically contains an originof replication, a promoter, as well as specific genes which allowphenotypic selection of the transformed cells. Vectors suitable for usein the present invention include, but are not limited to, the T7-basedexpression vector for expression in bacteria (Rosenberg, et al., Gene,56:125, 1987), the pMSXND expression vector for expression in mammaliancells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, metallothionein I, orpolyhedrin promoters).

Polynucleotide sequences encoding TfR2 can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art. Such vectorsare used to incorporate DNA sequences of the invention.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques that are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested after anexponential growth phase and subsequently treated by the CaCl₂ method,using procedures well known in the art. Alternatively, MgCl₂ or RBC1 canbe used. Transformation can also be performed after forming a protoplastof the host cell, if desired.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also beco-transformed with DNA sequences encoding TfR2 of the invention, and asecond foreign DNA molecule encoding a selectable phenotype, such as theherpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein. (See, for example, Eukaryotic Viral Vectors, ColdSpring Harbor Laboratory, Gluzman ed.k 1982).

Isolation and purification of microbial-expressed polypeptides, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

Another aspect of the present invention provides isolated TfR2polypeptide, or fragments thereof, and functional equivalents thereof.As used herein, the term “isolated” means a protein molecule, free ofcellular components, and/or contaminants normally associated with anative in vivo environment. The polypeptides of the present inventioninclude any isolated naturally occurring allelic variant, as well asrecombinant forms thereof.

Minor modifications of the primary amino acid of the peptide of thepresent invention may result in peptides which have substantiallyequivalent activity as compared with the specific peptide describedherein. Such modifications may be deliberate, as by site-directedmutagenesis, or may be spontaneous. Modification may also be made to thelength of the peptide of the present invention. It is recognized bythose skilled in the art that it is possible that a peptide which islonger or shorter than the peptide of the present invention may stillpreserve substantially the same biological function of the peptide ofthe present invention. All of the peptides produced by thesemodifications are included herein as long as the biological activity ofthe peptides still exists.

The polypeptide of the present invention can be isolated using variousmethods well known to a person of skill in the art. The methodsavailable for the isolation and purification of the polypeptides of thepresent invention include precipitation, gel filtration, ion-exchange,reverse-phase and affinity chromatography. Other well-known methods aredescribed in Deutscher et al., Guide to Protein Purification: Methods inEnzymology, Vol. 182 (Academic Press, (1990)), which is incorporatedherein by reference. Alternatively, the isolated polypeptides of thepresent invention can be obtained using well-known recombinant methodsas described, for example, in the Examples.

An example of the means for preparing the invention polypeptides is toexpress nucleic acids encoding the TfR2 in a suitable host cell asdescribed above. Polypeptides of the present invention can be isolateddirectly from cells that have been transformed with expression vectors.The polypeptide, biologically active fragments, and functionalequivalents thereof can also be produced by chemical synthesis. Forexample, synthetic polypeptides can be produced using AppliedBiosystems, Inc. Model 430A or 431A automatic peptide synthesizer(Foster City, Calif.), employing the chemistry provided by themanufacturer.

As used herein, the phrase “TfR2” refers to substantially pure nativeTfR2 proteins, or recombinantly expressed/produced proteins, includingvariants thereof encoded by mRNA, and generated by alternative splicingof a primary transcript, and further including fragments thereof whichretain native biological activity. Preferred polypeptides of the presentinvention are those that contain substantially the same amino acidsequence set forth in SEQ ID NO:1 (FIG. 7). In accordance with oneembodiment of the present invention, the isolated TfR2 polypeptide ofthe present invention is encoded by at least nucleotides set forth inSEQ ID NO:2 or SEQ ID NO:3 (See, FIG. 8 and FIG. 9, respectively). Inaccordance with one embodiment of the present invention, the sizes ofthe FLAG-tagged TfR2-α proteins are about 105 kDa in reducing condition,and about 215 kDa in non-reducing condition. The polypeptides of thepresent invention may be used to isolate ligands for transferrinreceptors.

A further aspect of the present invention provides antibodies which areimmunoreactive or bind to the peptides of the present invention.Antibodies which consist essentially of pooled monoclonal antibodieswith different epitopic specificities, as well as distinct monoclonalantibody preparations, are provided. Monoclonal antibodies are made fromantigen-containing peptides of the present invention or fragments bymethods well known in the art (Kohler, et al., Nature, 256:495, 1975;Current Protocols in Molecular Biology, Ausubel et al., ed., 1989).

Antibodies which bind to the peptides of the present invention or aregion of TfR2 represented by the peptides of the present invention canbe prepared using an intact polypeptide or fragments containing peptidesof interest as the immunizing antigen. A polypeptide or a peptide, suchas Sequence ID No.1, used to immunize an animal can be derived fromtranslated cDNA or chemical synthesis and is purified and conjugated toa carrier protein, if desired. Such commonly used carriers which arechemically coupled to the peptide include keyhole limpet hemocyanin(KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.The coupled peptide is then used to immunize the animal (e.g., a mouse,a rat, or a rabbit).

If desired, polyclonal antibodies can be further purified, for example,by binding to and eluting from a matrix to which a polypeptide or apeptide to which the antibodies were raised is bound. Those of skill inthe art will know of various techniques common in the immunology artsfor purification and/or concentration of polyclonal antibodies, as wellas monoclonal antibodies. (See, for example, Coligan, et al., Unit 9,Current Protocols In Immunology, Wiley Interscience, 1991, incorporatedby reference.)

The term “antibody” as used in this invention includes intact moleculesas well as fragments thereof, such as Fab, Fab′₂ and Fv, which arecapable of binding the epitopic determinant. These antibody fragmentsretain some ability to selectively bind with their antigen or receptorand are defined as follows:

-   -   (1) Fab, the fragment which contains a monovalent        antigen-binding fragment of an antibody molecule, can be        produced by digestion of a whole antibody with the enzyme papain        to yield an intact light chain and a portion of one heavy chain;    -   (2) Fab′₂, the fragment of an antibody molecule, can be obtained        by treating a whole antibody with pepsin, followed by reduction,        to yield an intact light chain and a portion of the heavy chain;        two Fab′ fragments are obtained per antibody molecule;    -   (3) Fab′2, the fragment of the antibody that can be obtained by        treating the whole antibody with the enzyme pepsin without        subsequent reduction; Fab′₂ is a dimmer of two Fab′ fragments        held together by two disulfide bonds;    -   (4) Fv, defined as a genetically engineered fragment containing        the variable region of the light chain and the variable region        of the heavy chain expressed as two chains; and    -   (5) Single chain antibody (“SCA”), defined as a genetically        engineered molecule containing the variable region of the light        chain, the variable region of the heavy chain, linked by a        suitable polypeptide linker as a genetically fused single chain        molecule.

Methods of making these fragments are known in the art. (See, forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988), incorporated herein by reference.)

As used in this invention, the term “determinant” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules, such as amino acids or sugar side chains, andusually have specific three-dimensional structural characteristics, aswell as specific charge characteristics.

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image’ of the epitope bound by the first monoclonal antibody. Thus, inthe present invention, an anti-idiotype antibody produced from anantibody which binds to, for example, the synthetic peptide of SequenceID NO.1, can act as a competitive inhibitor for site on TfR2 which isrequired for iron metabolism in cells.

The antibodies of the present invention can be used to isolate thepolypeptides of the present invention. Additionally, the antibodies areuseful for detecting the presence of polypeptides of the presentinvention, as well as analysis of chromosome localization, andstructural as well as functional domains.

Accordingly, another aspect of the present invention provides methodsfor detecting the presence of polypeptides of the present invention onthe surface of a cell. The method comprises contacting the cell with anantibody that specifically binds to TfR2 polypeptides, under conditionspermitting binding of the antibody to the polypeptides, detecting thepresence of the antibody bound to the cell, and thereby detecting thepresence of TfR2 polypeptides of the present invention on the surface ofthe cell. With respect to the detection of such polypeptides, theantibodies can be used for in vitro diagnostics or in vivo imagingmethods.

Immunological procedures useful for in vitro detection of target TfR2polypeptides in a sample include immunoassays that employ a detectableantibody; such immunoassays include, for example, ELISA, Pandexmicrofluorimetric assay, agglutination assays, flow cytometry, serumdiagnostic assays and immunohistochemical staining procedures which arewell known in the art. An antibody can be made detectable by variousmeans well-known in the art. For example, a detectable marker can bedirectly or indirectly attached to the antibody; useful markers include,for example, radionucleotides, enzymes, fluorogens, chromogens andchemiluminescent labels.

Furthermore, the antibodies of the present invention can be used tomodulate the activity of the TfR2 polypeptide in living animals, inhumans, or in biological tissues or fluids isolated therefrom.Accordingly, the present invention provides compositions comprising acarrier and an amount of an antibody having specificity for TfR2polypeptides effective to block binding of naturally occurring ligandsto TfR2 polypeptides.

Another aspect of the present invention provides an antisenseoligonucleotide capable of specifically binding to any portion of anmRNA that encodes TfR2 polypeptides so as to prevent or inhibittranslation of the mRNA and inhibiting the translation of mRNA of TfR2polypeptides. The antisense oligonucleotide may have a sequence capableof binding specifically with any portion of the sequence of the cDNAencoding TfR2 polypeptides. As used herein, the phrase “bindingspecifically” encompasses the ability of a nucleic acid sequence to formdouble-helical segments therewith via the formation of hydrogen bondsbetween the complementary base pairs. An example of an antisenseoligonucleotide is an antisense oligonucleotide comprising chemicalanalogs of nucleotides.

In accordance with the present invention, it is provided compositionscomprising an amount of the antisense oligonucleotide, described above,effective to reduce expression of TfR2 polypeptides by passing through acell membrane and binding specifically with mRNA encoding TfR2polypeptides so as to prevent translation and an acceptable hydrophobiccarrier capable of passing through a cell membrane. Antisenseoligonucleotide compositions are useful to inhibit translation of mRNAencoding TfR2 polypeptides. In accordance with one embodiment of thepresent invention, kits comprising the antisense of the presentinvention are also provided for inhibiting the expression of TfR2polypeptides. In accordance with another embodiment of the presentinvention, the compositions may be used to modulate levels of expressionof TfR2 polypeptides.

The present invention also provides compositions containing anacceptable carrier and any isolated, purified TfR2 polypeptide, anactive fragment thereof, or a purified, mature protein and activefragments thereof, alone or in combination with each other. Thesepolypeptides or proteins can be recombinantly derived, chemicallysynthesized or purified from native sources. As used herein, the term“acceptable carrier” encompasses any of the standard pharmaceuticalcarriers, such as phosphate buffered saline solution, water andemulsions such as an oil/water or water/oil emulsion, and various typesof wetting agents.

EXAMPLES Experimental Procedures

Cell Lines. HL-60, KG-1, U937 (myeloid leukemia); TF-1, K562 (erythroidleukemia); Jurkat, Molt-4 (T cell leukemia); Raji (Burkitt's lymphoma);LNCaP, PC-3 (prostate cancer); MCF-7, MDA-MB-231 (breast cancer); IMR-32(neuroblastoma); SK-Hep1 (hepatoma); HepG2 (hepatoblastoma); U-2OS(osteosarcoma) and SW480 (colon cancer) cell lines were obtained fromAmerican Type Culture Collection (ATCC, Manassas, Va.). ML-1, NB4 andKasumi 3 (myeloid leukemia), and both CHO-TRVb (TfR deficient Chinesehamster ovary) and TRVb-1 (human TfR stably transfected TRVb) cells werekindly provided by Drs. Minowada (14), Lanotte (15), Asou (16) andMcGraw (17), respectively. Human mononuclear cells were isolated fromthe blood of a normal volunteer by centrifugation on a Ficoll-Paque(Pharmacia, Piscataway, N.J.) gradient at 400×g for 30 min. Informedconsent was obtained from the individual.

Molecular Cloning of cDNA and genomic DNA. Complementary DNA librarieswere constructed from TF-1 and HL60 cells using a commercial kit(Marathon cDNA Amplification Kit, Clontech, Palo Alto, Calif.) and wereused for 5′- and 3′-RACE reactions to obtain a full-length cDNA clone.Primers A and B (see Table 1) were used for 5′- and 3′-RACE,respectively. The products of RACE reactions were subcloned into thepGEM-Teasy vector (Promega, Madison, Wis.). We isolated two transcriptsof 2.9 (α) and 2.5 (β) kb from the TF-1 and HL60 cDNA libraries,respectively. TABLE I Primer Sequences for TfR2 These primers were usedto amplify the TfR2 cDNAs in the RACE and RT-PCR analyses. Locations ofthese primers are shown as the nucleotide numbers in theTfR2-α-transcript sequence (GenBank accession number AF067864). Also,the locations of primers A, C, D and E are shown in FIG. 2. Primer NameSequence Direction Location A 5′-CCACACGTGGTCCAGCTTCTGGCGGGAG-3′ Reverse603-576 B 5′-CAGTTGCATCATCAGGCCTTCC-3′ Forward 1,061-1,082 C5′-ACGTCTCTGGCATCCTTCC-3′ Forward TfR2-β only D5′-GTGGTCAGTGAGGATGTCAA-3′ Forward 376-395 E 5′-TGTAGGGGCAGTAGACGTCA-3′Reverse 733-714

Genomic DNA was isolated from a human genomic library (Lambda FIX IILibrary, Stratagene, La Jolla, Calif.) using a 2.2 kbp fragment of the3′-end of the TfR2 cDNA as a probe (shown as probe-1 in FIG. 1). Afterrestriction enzyme mapping, a 3.85 kb fragment which included exons 4-6was subcloned into the pBluescript II(+) plasmid (Stratagene) (FIG. 1).Complementary and genomic DNA sequences were determined using an ABIPrism 373 automated sequencer (Perkin-Elmer, Foster City, Calif.).

Chromosomal Mapping. The GeneBridge 4 Radiation Hybrid Panel, RHO2(Research Genetics, Huntsville, Ala.) was used to determine thechromosomal location of the TfR2 gene as previously described (18). Theprimers A and C amplified a 178 bp fragment located in exon 4 (FIG. 2).The PCR products were electrophoresed through a 1.5% agarose gel,Southern blotted and hybridized with a ³²P-labeled probe of TfR2 (1 kbpfragment of the 5′-portion of the β form cDNA; shown as probe-2 inFIG. 1) to identify the hybrid clones containing the gene. The resultswere analyzed by accessing the database at the web sitehttp://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl.

Northern Blot and RT-PCR Analyses. Northern blot and RT-PCR analyseswere performed as previously described (18) with some modification.Human tissue Northern blot membranes and cDNAs were purchased fromOriGene (Rockville, Md.). For Northern blot analysis, two TfR2 cDNAfragments (probe-1 and -2 as shown in FIG. 1), a human β-actin cDNAfragment (OriGene) and an approximately 300 bp TfR cDNA fragment wereused as probes. For RT-PCR, the a form-specific primers (primers-A and-D) and the β form-specific primers (primers-C and -E) were used (Table1 and FIG. 2). Conditions for amplification were 35 cycles of 94° C. for30 s, 56° C. for 40 s and 72° C. for 1 min. As a control,glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was amplified in aseparate reaction using primers, 5′-CCATGGAGAAGGCTGGGG-3′ and5′-CAAAGTTGTCATGGATGACC-3′ for 27 cycles. The product waselectrophoresed through a 1.5% agarose gel, transferred to nylonmembranes, hybridized with radiolabeled TfR2 and G3PDH probes andautoradiographed.

Transfection and Immunoblotting. CHO-TRVb cells were maintained inF12-nutrient mixture (Gibco-BRL) supplemented with 5% fetal bovineserum. An amino terminal FLAG-tagged TfR2-α cDNA was subcloned intopcDNA3 (Invitrogen, Carlsbad, Calif.). This plasmid (100 μg) wastransfected into CHO-TRVb cells using Lipofectin (Gibco-BRL). Fortransient expression, cells were harvested 48 h after the transfection.We also isolated a stably expressing clone using G418 (200 μg/ml)selection and a standard limiting dilution method. The proteinexpression was confirmed by immunoblotting using anti-FLAG (M5) antibody(Eastman Kodak, New Haven, Conn.). Immunoblot analysis was performed aspreviously described (19).

Flow Cytometric Analysis of Tf-binding to the Cell Surface.Approximately 3×10⁵ cells were incubated with 5 μg/ml of biotinylatedhuman holo-Tf (Sigma) in 500 μl MEM α media (GIBCO) either in thepresence or absence of nonlabeled human holo-Tf (Sigma) or human Lf(Calbiochem, San Diego, Calif.) for 30 min on ice. After two washes withPBS supplemented with 0.1% bovine serum albumin, the cells wereincubated with streptavidin-PE (DAKO). The cells were washed twice againand were subsequently analyzed by flow cytometry.

Analysis of Tf-mediated Iron Uptake. One milligram of human apo-Tf(Sigma) in 0.5 ml of 0.25 M Tris-HCl, 10 μM NaHCO3, pH 8.0 was mixedwith 0.5 ml of 100 mM disodium nitrilotriacetate containing 0.4 mCi⁵⁵FeCl₃ (NEM, Boston, Mass.). The mixture was incubated at roomtemperature for 1 h and radiolabeled Tf was separated by filtration on aPD-10 column (Pharmacia). A specific activity of 27,000 cpm/μg wasobtained. Cells were incubated with ⁵⁵Fe-Tf in MEMα media in thepresence or absence of 200-fold excess of nonlabeled holo-Tf at 37° C.with 5% CO₂. After washing with PBS, the cells were lysed with 0.1 NNaOH and the radioactivity was counted using a liquid scintillationcounter.

Results

Molecular Cloning, Chromosomal Mapping and the Genomic Structure of theTfR2 Gene. We isolated seventeen 5′-RACE clones and ten 3′-RACE clonesfrom the 7F-1 cDNA library. Assembly of their nucleotide sequencesindicate an approximately 2.9 kb cDNA sequence (a form; GenBankaccession number AF067864). Using 5′ and 3′ gene-specific primers, acDNA clone encompassing the putative full-length coding sequence wascreated by PCR from the TF-1 cDNA library. This indicated that thepredicted cDNA sequence belonged to an actual expressed mRNA. When weused a HL60 cDNA library for cloning TfR2, the 5′-RACE products wereshorter than those from the TF-1 library, and the sequences around the5′-end were different (β form). All 5′-RACE products from the TF-1library belonged to the a form, and all 5′-RACE products from HL60belonged to the β form.

FIG. 1 shows the map of the TfR2 gene. According to FIG. 1, anapproximately 16 kbp genomic fragment was cloned from a human genomiclibrary (genomic clone 1) and restriction enzyme sites were mapped. A3.85 kbp fragment of the genomic clone 1 (shown as a shaded bar) wassubcloned into the pBluescript II(+) plasmid and sequenced. Theexon-intron borders shown in this figure were based on data deposited inthe GenBank (accession number AP053356) with some modifications based onour data. The α transcript contains 18 exons (closed boxes on the line).The β transcript lacks exons 1-3, and has an additional 142 bases at the5′-end of exon 4 (an open box on the line). The lower two boxes are thestructures of the α and β transcripts. IC, TM and EC indicate thesequences encoding intracellular, transmembrane and extracellulardomains, respectively. The locations of the probes that were used in thepresent invention are shown under the boxes.

According to the radiation hybrid panel analysis, TfR2 mapped onchromosome 7q22, between the D7S651 and WI-5853 markers. The restrictionenzyme mapping and partial sequencing of a 16 kb genomic DNA clone andcomparison with the deposited unpublished genomic sequence (20) revealedthat the α form consisted of 18 exons (FIG. 1). However, somedifferences between their exon-intron borders and ours were noted. OurDNA sequence of the TfR2-α transcript contained an additional 81nucleotides in exon 8 (nucleotides 1,053-1,133 in the TfR2-α transcript;GenBank accession number AF067864) and lacked 18 nucleotides in exon 18(between nucleotides 2,163 and 2,164) as compared with their predictedmRNA sequence (20). This resulted in a twenty-seven amino acid additionand a six amino acid deletion for our predicted TfR2-α protein. Also,our mRNA sequence contained an additional 298 nucleotides in the3′-untranslated region (UTR) (nucleotide 2580 to 2877).

The β form, which may be an alternative product of splicing or promoterusage, lacked exons 1, 2 and 3, and its first exon (exon 4 of the αform) had an additional 142 nucleotide bases at the 5′-end (FIGS. 1 and2). FIG. 2 shows the DNA sequences of exons 3-5. Boxed sequences werefound only in the β transcript. Arrows with solid and broken linesindicate the primer sequences used to synthesize the α and βtranscripts, respectively, by RT-PCR. Putative translation initiationcodon for the β transcript is shown as bold “ATG”. Guanines at −3 and+4, which are consistent with Kozak's sequence for this initiationcodon, are underlined.

The Primary Structure of TfR2 Proteins and mRNAs. The predicted aminoacid sequence of TfR2-α is shown in FIG. 3. FIG. 3 shows the deducedamino acid sequence of TfR2-α-aligned with those for the human TfR andPSMA proteins. Identical residues are boxed. Hydrophobic amino acidstretches located in the putative transmembrane portions are shaded. Theinternalization motif of TfR and the correspondingly similar motif ofTfR2-α are double underlined. Predicted initial methionine of TfR2-β isshown as a bold letter.

The hydrophobic stretch of residues from 81 to 104 following a pair ofarginines represents the predicted transmembrane domain. It is locatedclose to the amino terminus, similar to the transmembrane domains of TfRand PSMA (shaded section in FIG. 3) (10, 21). By analogy to TfR andPSMA, TfR2-α probably is a type II membrane protein. Therefore, residues1 to 80 of TfR2-α may be the cytoplasmic domain and residues 105 to 801the extracellular domain. In the extracellular domain, amino acidsequence homologies between TfR2-α and either TfR or PSMA were quitehigh. The extracellular domain of TfR2-α was 45% identical and 66%similar with that of TfR. With PSMA, the identity was 27% and thesimilarity 60%. The cysteine residues at positions 89 and 98 of TfR formdisulfide bonds resulting in homodimerization. Two cysteine residues atpositions 108 and 111 in TfR2-α are located in an analogous region andmay serve a similar function. In addition, TfR2-α contains the motifYQRV (amino acid 23-26) in the middle of the cytoplasmic domain, thatmay function as an internalization signal, similar to the YTRF motif inTfR (FIG. 3, double underlined) (22-24).

The β transcript lacks exons 1 to 3, which encode the entiretransmembrane and cytoplasmic domains as well as part of theextracellular domain including the two cysteine residues at 108 and 111.The additional 142 nucleotide 5′-sequence in exon 4 does not contain aninitiation codon. Translation probably starts at the ATG located atnucleotide 542, which is in frame with the α transcript ORF. Thepredicted initial methionine is shown in FIG. 2, exon 4 and FIG. 3 asbold “ATG” and “M”, respectively. This ATG contains a G at positions −3and +4 indicating it is an ideal start site for translation (25). Hencethe predicted protein product of the β transcript would lack both atransmembrane domain and signal peptide, resulting in a possibleintracellular protein that may or may not be functional.

Although the primary structure of the TfR2-α protein seemed to be quitesimilar to TfR, the 3′-UTR of the TfR2 mRNA was shorter than that of theTfR transcript. Also, a typical iron-responsive element (IRE) was notpresent in the UTRs of either of the TfR2 transcripts (26).

Characterization of TfR2 mRNA Expression. FIGS. 4A and 4B show theresults of the Northern blot analysis of poly A+ RNA from human tissues.Hybridization was with ³²P-labeled TfR2 probes. FIG. 4A shows multipletissue blots of human mRNA were hybridized with a TfR2 probe (probe No.1 in FIG. 1). Membranes were hybridized in the same bottle at the sametime, and the autoradiograms were developed after a 12 hr exposure. InFIG. 4B, thirty micrograms of total RNA from cell lines were loaded ineach lane and hybridized with a TfR2 probe (probe No. 2) and a TfRprobe. A ³²P-labeled β-actin probe was used as a control for all blots.Molecular weight markers or the positions of ribosomal RNA are indicatedon the left.

Northern blot analysis of poly A+ RNA from human tissues showed that a2.9 kb mRNA for TfR2 was expressed predominantly in the liver and, to alesser degree, in the stomach (FIG. 4A). This corresponded with thelength of TfR2-α cDNA isolated from TF-1 cells. In addition, faint bandsat 4 kb (stomach) and 1.7 kb (liver, lung, small intestine, stomach,testis and placenta) were observed. These bands may reflect the presenceof additional alternative forms of TfR2 mRNA. Northern blot analysis oftotal RNA of various cell lines revealed a high expression of TfR2-α inK562 (erythroleukemia) and HepG2 (hepatoblastoma) (FIG. 4B). Theexpression levels of TfR2-α were not always correlated with those of TfR(FIG. 4B). No transcripts corresponding to TfR2-β (2.5 kb) were observedby Northern blot analysis.

To compare the expression of the α and β transcripts, RT-PCR wasperformed using specific primers for each form. FIGS. 5A and 5B show therepresentative results of RT-PCR analyses. RT-PCRs were performed withprimers for α and β transcripts of TfR2 (35 cycles) as well as G3PDH (27cycles). The products were electrophoresed through 1.5% agarose gels,transferred to nylon membranes, hybridized with radiolabeled probes andautoradiographed. FIG. 5A shows cDNA panels of human tissues. (MNC;human peripheral blood mononuclear cells.) FIG. 5B shows cDNAs fromvarious human cell lines. Experiments were repeated at least twice foreach sample, and the figures are representative results. The cDNAs forML-1, Kasumi-3, HL60 and MDA-MB-231 are negative, but showed tracelevels of α form expression in other experiments.

FIGS. 5A and 5B show that using a human tissue cDNA panel as a template,the expression of the α form was limited to the liver, spleen, lung,muscle, prostate and peripheral blood mononuclear cells (FIG. 5A). Onthe other hand, expression of the β form occurred in all of the humantissues tested. Human cancer cell lines from various tissues werestudied for expression of the two transcripts. Most of the cell linesexpressed both transcripts except three; SK-Hep1 (hepatoma) lacked bothα and β transcripts, HepG-2 (hepatoblastoma) and ML-1 (myeloblast)lacked the β transcript (FIG. 5B). Neither deletion nor rearrangement ofthe TfR2 gene was detected in Southern blot analysis in SK-Hep1 (datanot shown).

Tf-binding to the TfR2-α Transfected Cells. To analyze the function ofTfR2-α, we stably transfected CHO-TRVb cells, which lack functional TfR,with FLAG-tagged TfR2-α. FIGS. 6A, 6B and 6C show the expression andfunctional analysis of TfR2-α protein. In FIG. 6A, Tf-binding to thecell surface was examined in neomycin resistant control CHO-TRVb cells(left panels), FLAG-tagged TfR2-α stably transfected cells (middle) andTRVb-1, TfR stably transfected cells (right). The cells were incubatedwith 5 μg/ml of biotinylated human holo-Tf in MEM α media for 30 min onice. After washing with PBS, the cells were incubated withstreptavidin-PE, and analyzed by flow cytometry. The solid lines showthe histograms without competition. Competition experiments wereperformed in the presence of either 10-fold (- - - - ) or 100-fold (— -— - —) excess of either nonlabeled Tf (upper panels) or Lf (lowerpanels). In FIG. 6B, Tf-mediated ⁵⁵Fe uptake was examined in neomycinresistant control CHO-TRVb cells (Neo cells), human TfR stablytransfected cells (TfR cells) and FLAG-tagged TfR2-α stably transfectedcells (TfR2 cells). Closed symbols (-C) represent cold competitionexperiments with 200-fold excess of nonlabeled Tf. The mean±S. D. fromeither quadruplicate (without competition) or triplicate (coldcompetition) experiments is shown. In FIG. 6C, cell lysates from pcDNA3transiently transfected cells (lane 1) and FLAG-tagged TfR2 transfectedcells (lanes 2 and 3) were electrophoresed through a 4-15% lineargradient SDS-polyacrylamide gel. For the sample in lane3,2-mercaptoethanol was omitted from the sample buffer. Aftertransferring to a PVDF membrane, FLAG-fusion proteins were detected byimmunoblotting. The positions of molecular weight markers are indicatedon the left.

In FIGS. 6A, 6B and 6C, the cell surface Tf-binding was examined usingbiotinylated Tf and flow cytometry. Neomycin resistant control cellswere almost negative for the cell surface Tf-binding (FIG. 6A, left).TRVb-1, the human TfR stably transfected cells were positive for cellsurface binding of Tf, and this binding was competed by nonlabeled Tfbut not by Lf (FIG. 6A, middle). For the CHO-TRVb cells stablyexpressing TfR2-α, the mean level of cell surface Tf-binding was clearlyhigher than that of the control cells (FIG. 6A, right, solid lines). Incompetition experiments, 10-fold excess of nonlabeled Tf markedlyinhibited the binding of biotinylated Tf, but even 100-fold excess of Lfdid not inhibit the binding (FIG. 6A, right, broken lines). Tf-bindingto the TfR2-α cells was also examined in a transient expression systemusing CHO-TRVb cells, and the levels of Tf-binding to the cell surfacewere consistently as follows: TfR cells>TfR2-α cells>pcDNA3 cells (datanot shown).

Tf-mediated ⁵⁵Fe Uptake of theTfR2-α-Transfected Cells. Human TfR andTfR2-α stably transfected CHO-TRVb cells were incubated with ⁵⁵Fe-Tf,and ⁵⁵Fe uptake was measured. Neomycin resistant CHO-TRVb cells wereused as controls. Tf-mediated ⁵⁵Fe uptake by the TfR2-α cells wascomparable to TfR cells; both were clearly higher than control cells(FIG. 6B). Competition by 200-fold excess of nonlabeled Tf almostcompletely blocked ⁵⁵Fe incorporation in these three cell lines after a5 h incubation (FIG. 6B). In spite of the absence of functional TfR, aslight uptake of Tf-mediated ⁵⁵Fe was also observed in the control TRVbcells as previously reported by Chan, et al. (27).

Dimerization of the FLAG-tagged TfR2-α Proteins Expressed in MammalianCells. Cell lysates from the cells transiently transfected with pcDNA3empty vector or the FLAG-tagged TfR2-α plasmid were examined byimmunoblotting using anti-FLAG antibody (FIG. 6C). Two closely migratedbands of ˜105 kDa were observed in the cell lysate transfected withFLAG-tagged TfR2-α under reducing conditions (lane 2). When2-mercaptoethanol was omitted from the sample loading buffer, thedoublet of ˜105 kDa decreased, but a protein of ˜215 kDa appeared (lane3). Faint bands of ˜260 kDa and ˜125 kDa were also seen undernon-reducing conditions (lane 3, arrows).

Discussion

The primary structure of the TfR2-α protein deduced from its mRNA issimilar to that of TfR (see RESULTS). In addition, TfR2-α transfectedcells showed increases of both Tf-binding and Tf-mediated iron uptake(FIGS. 6A and B). However, the mechanisms that regulate expression ofTfR2 and TfR may be different. Levels of the TfR protein are regulatedpost-transcriptionally through IREs in its 3′-UTR, to which ironregulatory protein-I (IRP-1) and IRP-2 can bind. In cells lackingsufficient iron, IRPs bind to the iron-responsive elements of TfR mRNAand stabilize these transcripts. In the presence of excess intracellulariron, IRPs are released, leading to degradation of the TfR mRNA. Inrapidly growing cells, proto-oncogene c-MYC represses H-ferritin andupregulated IRP-2, and the upregulation of IRP-2 may increase TfRprotein expression (28). Neither the 3′- nor the 5′-UTRs of the TfR2mRNAs have a detectable IRE-like structure, suggesting anothermechanism(s) may regulate TfR2 expression.

Northern blot analysis using normal human poly A⁺ RNA from a variety oftissues showed that the liver was the only cell type that prominentlyexpressed TfR2-α (FIG. 4A). Also, TfR2-α was expressed highly in theK562 erythroleukemic cell line which is capable of hemoglobin synthesis(FIG. 4B). This result suggests that erythroid hematopoietic cells mayalso express high levels of TfR2-α. The major product of red blood cellsis hemoglobin which contains abundant iron, and if TfR2-α is involved iniron transport, it would be expected to be strongly expressed on thesecells. In erythroid cells, Cotner et al. predicted the presence of analternative form of TfR using a set of monoclonal antibodies against TfR(29). Their findings may be ascribed to TfR2-α.

The size of the FLAG-tagged TfR2-α expressed in mammalian cells is ˜105kDa in the presence of a reducing agent, and is ˜215 kDa in the absenceof a reducing agent (FIG. 6C), indicating dimerization of TfR2-α throughdisulfide bonds. The size of FLAG-tagged TfR2-α monomer, ˜105 kDa, islarger than the molecular weight calculated from the amino acid sequence(˜90 kDa). This may reflect post-translational modifications of theprotein such as glycosylation. Actually there are 4 putativeN-glycosylation sites (amino acids 240-243, 339-342, 540-543 and754-757) in the TfR2-α protein. Hence, the double bands of ˜105 kDa seenin FIG. 6C may be due to different states of glycosylation. In addition,faint bands of ˜260 kDa and ˜125 kDa just above the clear bands of ˜215kDa and ˜105 kDa, respectively, were observed under non-reducingconditions (FIG. 6C, lane 3, arrows). These faint bands may reflectinteraction of TfR2-α with a small protein (˜20 kDa) through disulfidebonds, which may or may not be a ligand.

To investigate the function of TfR2, Tf and other Tf family members wereconsidered as candidate ligands of TfR2. Six members of Tf family havebeen cloned to date; Tf, Lf, melanotransferrin (30), ovotransferrin,saxiphilin (31), and porcine inhibitor of carbonic anhydrase (32). Thelast two do not possess iron-binding properties and the last three havenot been identified in humans. Melanotransferrin is an unlikely TfR2ligand because it is a membrane-bound protein of melanoma cells. Only Tfand Lf remained as candidates. The CHO-TRVb cells transfected withFLAG-tagged TfR2-α showed higher levels of Tf-binding to the cellsurface than did the control cells (FIG. 6A). This indicates thatFLAG-tagged TfR2-α was expressed on the cell surface and was bound byTf. This binding was effectively competed by nonlabeled Tf but not by Lf(FIG. 6A). This indicates that Tf can bind to TfR2-α more specificallythan can Lf. In addition, Tf-mediated iron uptake by TfR2-α transfectedcells was obviously higher than that of control cells (FIG. 6B).

However, if the only ligand for TfR2-α is Tf and the main function ofTfR2-α is cellular iron uptake, why do the cells have two differentreceptors for Tf? TfR2-α may simply be another transferrin receptor witha different affinity. Possibly, the fate of the Tf/TfR2-α complex on thecell surface may be different from that of the Tf/TfR complex. Theputative internalization motif of TfR2-α is not identical to that ofTfR, and even a minor difference of the internalization motif may resultin different destinations of the endosomes (24). Still, the possibilitythat TfR2-α has another specific ligand other than Tf remains. Recently,the field of iron metabolism has been markedly advanced by thediscoveries of HFE, mutations of which occur in most of the patientswith hereditary hemochromatosis (8, 9), and Nramp2, an intestinal irontransporter (33, 34). Does TfR2-α-a bind to HFE, which normally forms acomplex with TfR on the cell membrane? If it does, TfR2-α may affect thecellular iron uptake by chelating HFE. Can TfR2-α form a heterodimerwith TfR? This may also affect cellular iron uptake. Elucidation of theprecise role of TfR2 may provide an important step for clarifying themechanisms and the regulation of cellular iron uptake.

We cloned two different forms of transcripts from TfR2 gene, α and β.Two different transcripts are also expressed from the PSMA gene, anothermember of the TfR-like family. The shorter form of PSMA lacks the 5′-endencoding the transmembrane domain (35), similar to the β-form of TfR2.Nearly a 100-fold difference in the ratio of expression of the longerand the shorter forms of PSMA mRNA has been reported during progressionof prostate cancer, with the shorter form predominant in normal cellsand the longer form predominant in the cancer cells (36). Using theextremely sensitive RT-PCR method, we could distinguish expression ofthe (α and β forms of the TfR2 gene. Among normal tissues, theexpression of the α (longer) form was detected in the liver, spleen,lung, muscle, prostate and peripheral blood mononuclear cells (FIG. 5A).The β form was distributed more widely. Interestingly, the two celllines derived from the liver (SK-Hep1 and HepG2) lacked expression ofthe β form, whereas most cell lines from other tissues as well as normalliver expressed this shorter form (FIGS. 5A and 5B).

We mapped TfR2 to chromosome 7q22. Deletion or loss of heterozygosity ofthis chromosomal region has been reported in several malignant diseasesincluding myelodysplastic syndromes, acute myeloid leukemia, as well asbreast, ovarian and pancreatic cancers (37-41). It is speculated thatTfR2 mutations may occur in these cancers.

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1-20. (canceled)
 21. A method for detecting the presence of TfR2 protein on a cell surface comprising the steps of: (a) providing an antibody specific for TfR2 protein; (b) contacting the cell with the antibody under conditions that allow the binding of the antibody to the TfR2 protein of the cell, and (c) detecting the antibody bound to the cell.
 22. The method of claim 21, wherein the antibody is labeled with a detectable marker.
 23. The method of claim 22, wherein the detectable marker is selected from a group consisting of radionucleotides, enzymers, fluorogens, chromogens, and chemiluminescent labels. 